Fig. 6

Expression of two tenascin genes is affected by temperature.

In situ hybridization against tenascin-c (tnc) (A-H) and tenascin-w (tnw) (L-O) in 22-ss wild-type (WT) and s1pr2^as10 (as10) mutant embryos raised at 28.5°C or 22.5°C. Expression of tnc increased in the pharyngeal arches (white arrows in B and D) and the cells between the brain and eyes of embryos raised at 22.5°C, but was unaffected in the trunk somite region (E-H). (I) qRT-PCR revealed a trend towards increased expression of tnc mRNA in s1pr2^as10 mutant embryos raised at 22.5°C. (J) Knockdown of tnc expression with tnc-MO1 in s1pr2^as10 mutants increased the percentage of 26-ss embryos raised at 22.5°C with the Class III cardia bifida phenotype. (K) Injection of s1pr2^as10 mutant embryos raised at 28.5°C with human TNC protein partially rescued cardia bifida phenotypes at 24 hpf. (L-O) Decreased tnw expression in all tissues, including scattered epidermal cells in the head (black arrows in M and O) was observed in both 22-ss WT and s1pr2^as10 mutants raised at 22.5°C. (P) qRT-PCR was used to confirm that expression of tnw mRNA is significantly reduced in 22-ss WT and s1pr2^as10 mutant embryos raised at 22.5°C. (Q) Knockdown of tnw with tnw-MO1 in s1pr2^as10 mutant embryos raised at 28.5°C partially rescued cardia bifida phenotypes at 24 hpf. (R) Injection of s1pr2^as10 mutant embryos raised at 22.5°C with 100 pg tnw mRNA increased the percentage of 26-ss embryos with the Class III cardia bifida phenotype. Scale bars = 100 μm. The error bars indicate the standard error. Statistical significance was determined using Student’s t-test. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.